Dr. Daniel Spade's long-term research goal is to improve male reproductive health by discovering the underlying mechanisms by which environmental stressors disrupt the development of the male reproductive system. Short-term objectives to reach this goal include developing rodent and human fetal testis model systems and describing the mechanisms of retinoic acid(RA) signaling disruption by phthalates in the developing fetal testis. This will provide the groundwork for experiments that will test the influence of phthalates on retinoic acid signaling leading to persistent later-life effects. Dr. Spade's plan for development toward an independent research career in male reproductive toxicology includes acquisition of new scientific skills in in vitro cell and tissue culture, pathoogy, and epigenomic analysis; career development training to include presentations at scientific meetings, publication of research results, leadership experience in the Society of Toxicology, mentoring of students, training in best teaching practices, and professional development workshop, as well as training in the Responsible Conduct of Research. This training plan will augment Dr. Spade's skills and abilities to support independent studies of these mechanisms of toxicity during the R00 phase and beyond. Environment Dr. Spade is a trainee in a supportive institutional environment in the Brown University Department of Pathology and Laboratory Medicine (PLM). His mentor for this K99 project is Kim Boekelheide, an expert reproductive toxicologist and experienced mentor of postdoctoral trainees. Dr. Boekelheide will serve on Dr. Spade's Internal Advisory Committee with Dr. Susan Huse and Dr. Richard Freiman, experts in computational biology and reproductive biology, respectively. The University and PLM provide excellent resources for trainee development in research, as well as professional development. Dr. Spade will have the support, facilities, and resources to successfully perform the histology, immunohistochemistry, microscopy, gene expression, and epigenome analyses for in vitro and in vivo experiments in this grant application. His professional development will be supported by his Internal Advisory Committee, PLM, and Brown's Office of Graduate and Postdoctoral Studies. Research on this project, Retinoic Acid Signaling Disruption by Phthalates in Human and Rodent Fetal Testis, will improve male reproductive health by providing mechanistic information about disruption of a critical fetal testis developmental pathway by a ubiquitous class of environmental chemicals. Retinoic acid signaling regulates the entry of testicular germ cells into meiosis, and exogenous retinoic acid has dramatic effects on germ cell and Sertoli cell proliferation, differentiation, and death. Phthalate treatment blocks signaling through retinoic acid receptor alpha in mouse Sertoli cells in vitro. Phthalates and retinoic acid both cause adverse effects on the fetal seminiferous cord of multiple species in vitro. However, the role of retinoic acid signaling disruption in the fetal testis response to phthalates is poorly understood. We have obtained preliminary evidence from coexposure experiments that phthalates and retinoic acid interact to produce significant adverse effects on fetal testis tissue in vitro. Durig the mentored phase, we will test mouse and rat in vitro organotypic fetal testis culture models to determine how Sertoli and germ cells respond to retinoic acid and phthalates, alone and in combination (Specific Aim 1). This will allow for important cross-species comparison with experiments in human fetal testis cultures, xenotransplants, and cultured primary Sertoli cells, which will begin during the mentored phase and continue into the independent phase (Specific Aim 2). After quantifying the response of mouse, rat, and human fetal testis histology, gene expression, control of transcription, and DNA methylation to phthalate-retinoic acid mixtures, additional experiments will measure the impact of fetal phthalate and retinoic acid exposure on the mouse testis in later life, including persistent impacts on DNA methylation. These experiments will occur during the independent phase (Specific Aim 3), and will serve as a first step toward identifying transgenerational effects of retinoic acid signaling disruption in the human germline. This project is guided by the working hypothesis: phthalates interfere with fetal testicular development through disruption of retinoic acid signaling in the seminiferous cord. The proposed experiments provide critical information about environmental exposures and effects on male reproductive health and serve the goal of this project: to describe the mechanism by which phthalates act on the retinoic acid signaling pathway to produce adverse outcomes during human fetal testicular development. This goal will be achieved by fulfilling the following Specific Aims: Aim 1: Determine how retinoic acid and phthalates impact rat and mouse fetal testis development [K99]. Aim 2: Quantify phthalate interaction with retinoic acid signaling in human fetal testis [K99/R00]. Aim 3: Identify persistent adverse outcomes of phthalate exposure mediated by altered retinoic acid signaling [R00].